Interpretive Summary: The vinegar flies Drosophila subobscura and D. obscura frequently serve as study organisms for evolutionary biology. However, accurate species identification of living specimens of both sexes is difficult, as the two species are morphologically very similar. In order to introduce wild-caught individuals to the laboratory with the aim to retain genetic variation, a rapid and non-destructive method for species identification with the potential for high throughput is needed as an alternative to morphology-based methods. Here we test the usefulness of the non-invasive method near-infrared (NIR) spectroscopy for discriminating live individuals of the two species. We found a classification success for wild-caught specimens of 85%, and the species specificity of the chemical profiles persists in laboratory offspring (87-92% success). This rapid NIR technique helped us to conclude that the cuticular chemistry is genetically determined, despite changes in the cuticular fingerprints which we interpret as due to laboratory adaptation, genetic drift and/or diet changes. Also, we demonstrated that by applying an appropriate cut-off value for interpreting the prediction values, the classification success can be immensely improved (to up to 99%), albeit at the cost of excluding a portion of specimens.

Technical Abstract:
The vinegar flies Drosophila subobscura and D. obscura frequently serve as study organisms for evolutionary biology. Their high morphological similarity renders traditional species determination difficult, especially when living specimens for setting up laboratory populations need to be identified. Here we test the usefulness of cuticular chemical profiles collected via the non-invasive method near-infrared spectroscopy for discriminating live individuals of the two species. We find a classification success for wild-caught specimens of 85%. The species specificity of the chemical profiles persists in laboratory offspring (87-92% success). Thus, we conclude that the cuticular chemistry is genetically determined, despite changes in the cuticular fingerprints which we interpret as due to laboratory adaptation, genetic drift and/or diet changes. However, because of these changes, laboratory-reared specimens should not be used to predict the species-membership of wild-caught individuals, and vice versa. Finally, we demonstrate that by applying an appropriate cut-off value for interpreting the prediction values, the classification success can be immensely improved (to up to 99%), albeit at the cost of excluding a considerable portion of specimens from identification.